CN102648275B - Phage phi 29DNA polysaccharase mosaic - Google Patents
Phage phi 29DNA polysaccharase mosaic Download PDFInfo
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- CN102648275B CN102648275B CN201080039946.9A CN201080039946A CN102648275B CN 102648275 B CN102648275 B CN 102648275B CN 201080039946 A CN201080039946 A CN 201080039946A CN 102648275 B CN102648275 B CN 102648275B
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- 150000002972 pentoses Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000037048 polymerization activity Effects 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
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Abstract
The invention belongs to biological technical field.Specifically, the present invention relates to a kind of archaeal dna polymerase mosaic and the purposes for copying template DNA, increasing or check order thereof, described mosaic comprises N-terminal (N-end) district of coding φ 29 type archaeal dna polymerase and C-terminal (C-end) district containing at least one HhH structural domain, and the two combines by connecting aminoacid sequence.Equally, the invention provides method and a kind of test kit for implementing described method that a kind of described archaeal dna polymerase mosaic copies thymus nucleic acid, increases or check order.
Description
The invention belongs to biological technical field.Specifically, the present invention relates to a kind of archaeal dna polymerase mosaic and the purposes for copying template DNA, increasing or check order thereof, described mosaic comprises N-terminal (N-end) district of coding φ 29 type archaeal dna polymerase and C-terminal (C-end) district containing at least one HhH structural domain, and the two combines by connecting aminoacid sequence.Equally, the invention provides a kind of use described archaeal dna polymerase mosaic to copy thymus nucleic acid, increase or check order method and a kind of test kit for implementing described method.
prior art level
The phage phi 29 unique enzyme copied required for its genome is its archaeal dna polymerase, and this can catalyze and synthesize again the monomeric protein of the 66KDa of chain extension by catalysis replication initiation.For initial, this polysaccharase be called the protein bound of " end " (TP), identify the end of φ 29DNA and catalysis TP-dAMP covalent complex is formed.After being polymerized 10 Nucleotide, archaeal dna polymerase/TP heterodimer has dissociated, and extends the chain from DNA.
Replicability archaeal dna polymerase needs and the accessory protein of combination between stabilized enzyme and DNA interacts (Kuriyan and O ' Donnell.JMolBiol.1993; 234:915-925).On the other hand, described archaeal dna polymerase must when the DNA chain that was not replicated at that time is separated coupling polymerization, their need to associate with the function of helicase type albumen for this reason.In this respect, the archaeal dna polymerase of phage phi 29 has the following various built-in function characteristics making its uniqueness:
A) high processivity (being defined through the nucleotide number that binding events mixes);
B) high chain separating power, this makes it can copy the genome of described phage when there is not helicase type accessory protein.Processivity is separated these two characteristics and makes φ 29DNA polysaccharase can synthesize more than long DNA chain (Blanco etc., the JBiolChem.1989 of 70kb with chain; 264:8935-8940);
C) in new chain, insert high accuracy (Esteban etc., the JBiolChem.1993 of Nucleotide; 268:2719-2726).
All these characteristics cause developing various isothermal process (constant temperature) scheme based on using this polymeric enzymatic amplification double-stranded DNA (dsDNA).In simple configuration, φ 29DNA polysaccharase utilizes the ability of single-stranded cyclic DNA (ssDNA) to make by rolling loop type method (or the amplification of RCA-rolling loop type) DNA amplification, produce very long more than 10 circular template that contain and copy ssDNA molecule (Blanco etc., JBiolChem.1989; 264:8935-8940; US5001050, US5198543 and US5576204).By AmershamBiosciences/MolecularStaging (Dean etc., GenomeRes.2001; 11:1095-1099; Dean etc., ProcNatlAcadSciUSA.2002; 99:5261-5266) develop for increasing in the method for dsDNA, φ 29DNA polysaccharase will be used to combine with use sexamer (six-Nucleotide) random-sequence primers, make it possible to from the pieck stage cyclic plasmid DNA [Templiphi of GEHealthcare
tM] or from the 10 nanogram genomic dna [Genomiphi of GEHealthcare
tMwith Qiagen's
] start to obtain 10
4-10
6amplification factor.Produce product there is high quality, and can directly digested or order-checking and at front purifying, need not this demonstrate that φ 29DNA polysaccharase is the strongest enzyme for this object.NaCl or KCl and the MgCl that add different concns (mmole level) for implementing to use the common damping fluid of the amplified reaction of φ 29DNA polysaccharase to contain tris-HCl (pH7.5)
2(US20030207267).But, although these schemes are satisfactory in several cases, but still day by day need to develop other scheme allowed from less DNA amount.
No matter how and with it DNA sequence dna combines HhH (" spiral-hair clip-spiral ") die body, and is present in (Shao and Grishin.NucleicAcidsRes.2000 in various archaeal dna polymerase, ligase enzyme and glycosylase; 28:2643-2650; Doherty etc., NucleicAcidsRes.1996; 24:2488-2497).These die bodys contain a pair antiparallel alpha-helix connected by " hair clip " type ring.Second alpha-helix is not outstanding from this structure, and therefore, do not resemble other DNA in conjunction with die body, it can not be inserted in the major groove of DNA.Crystallographic Study shows, sets up protein-DNA interact by " ring " between two alpha-helixs.This ring participates in setting up the non-specific interaction with DNA, and usually containing consensus sequence GhG, wherein h is hydrophobic residue, is generally I, V or L.Crystallographic structure is resolved and is shown, sets up and interact between the nitrogen of polypeptide chain and the oxygen of the phosphoric acid of DNA.In addition, set up other interactional polare Aminosaeren with phosphate and be often present in 2 and 3 relative to second G.The last G of consensus sequence forms the N-terminal portions of second alpha-helix, and hydrophobic residue h facilitates the interaction between two alpha-helixs of die body.Two alpha-helixs are packaged, form 25-50 ° of angle among each other, which dictates that distinctive hydrophobic pattern in sequence.HhH die body is usually formed and is called (HhH)
2the part of primary structure, (HhH)
2be made up of two the HhH die bodys combined by alpha-helix, about promotion, it becomes specular (Shao and Grishin.NucleicAcidsRes.2000 with the DNA of alpha-helix stable bond to these two die bodys; 28:2643-2650; Doherty etc., NucleicAcidsRes.1996; 24:2488-2497; Thayer etc., EMBOJ.1995; 14:4108-4120).
Previously already heat-stable DNA polymerase (such as Taq and Pfu) was formed mosaic for improving DNA binding ability by described polysaccharase between non-specific DNA is in conjunction with die body.(Pavlov etc., ProcNatlAcadSciUSA.2002; 99:13510-13515; WO2004013279; Wang etc., NucleicAcidsRes.2004; 32:1197-1207).
The crystal of φ 29DNA Polymerase Structure resolves the molecular basis to pre-polymerization providing and be responsible for chain separation coupling, and this is special feature (Kamtekar etc., 2006 of this enzyme; EMBOJ25:1335-1343).
The comparative analysis of carrying out to other eucaryon type archaeal dna polymerase (B family) shows similar General folding: pointed by general subdomain, C-end polymeric structural domain that palm and thumb are formed, and it forms passage, by this passage in conjunction with DNA; And be responsible for removing 3 '-5 ' N-end exonuclease domain of the Nucleotide that mistake is mixed between polymerization period.Major structural differences between the archaeal dna polymerase of known structure and φ 29DNA polysaccharase, it is the subdomain that existence two is other in the paradigmatic structure territory of the latter, these two subdomains all correspond to the insertion of conserved sequence in archaeal dna polymerase subgroup, and described insertion is with being called that the albumen of TPR1 and TPR2 is as primer.Subdomain TPR1 near palm, and with DNA double helical contact.Near subdomain thumb, palm and finger, the subdomain TPR2 of tool β-hairpin structure forms ring structure (it surrounds the DNA of new synthesis completely), and archaeal dna polymerase is combined with DNA, and it is for needed for the copying of forward mode.Equally, subdomain TPR2 points with subdomain, palm is together with exonuclease domain, participate in the formation (template strand arrives active centre by this throat between replicative phase) of throat, force double-stranded DNA separately along with polysaccharase moves, work with the similar manner worked with helicase, and ability (Kamtekar etc., 2006 with chain separation coupling will be polymerized for polysaccharase provides; EMBOJ25:1335-1343; Rodr í guez etc., 2005; ProcNatlAcadSciUSA102:6407-6412).φ 29DNA polysaccharase has such effect about this significant difference of all the other polysaccharases in paradigmatic structure territory, and namely peptide will have its unpredictalbe binding characteristic in the fusion of its C-end in polymerization and DNA.
invention summary
The present invention relates to a kind of archaeal dna polymerase mosaic and the purposes for copying template DNA, increasing or check order thereof, described mosaic comprises the N-end region of coding φ 29 type archaeal dna polymerase and the C-end region containing at least one HhH structural domain, and the two combines by connecting aminoacid sequence.Equally, the invention provides a kind of use described archaeal dna polymerase mosaic to copy thymus nucleic acid, increase or check order method and a kind of test kit for implementing described method.
Phage phi 29DNA polysaccharase has the very interesting several features for DNA amplification, such as: the high processivity participated in without the need to any accessory protein; With high chain separating power, this make its can with DNA single one binding events in copy the genome of described phage; And in new chain, insert the high accuracy of Nucleotide.These characteristics cause developing various of using the scheme for isothermal duplication DNA of this polysaccharase, and this polysaccharase makes it possible to obtain need just can by the high-quality product directly digested or check order at front purifying.But, still need to allow the scheme from its DNA amplification more in a small amount.The present invention responds this needs by two kinds of approach: 1) exploitation significantly improves the specificity of reaction and the composition of output; And 2) φ 29DNA polysaccharase and strengthen this enzyme the non-specific DNA of DNA binding ability in conjunction with die body between form mosaic.
Show in this patent embodiment, by polyoxyethylated sorbitan monolaurate (
20) and ammonium salt add in the damping fluid being generally used for increasing with φ 29DNA polysaccharase simultaneously, prevent non-specific DNA cloning on the one hand, make it possible to fly gram plasmid DNA of (fg) from limited amount 0.1 and realize detectable specific amplification as the 10fg genomic dna of template on the other hand.
For raising DNA binding ability, in the present invention the HhH die body of the topoisomerase V of Kan Deleshi methane thermophile bacteria (Methanopyruskandleri) is merged by the carboxyl terminal being derived from 4 kinds of chimeric catenation sequences of difference or joint and φ 29DNA polysaccharase.These mosaics can from the amount amplification template DNA be less than needed for natural φ 29DNA polysaccharase.
First aspect present invention relates to a kind of archaeal dna polymerase mosaic (hereinafter referred to as archaeal dna polymerase mosaic of the present invention), and described mosaic comprises:
A) by its C-end and the b) aminoacid sequence of coding φ 29 type archaeal dna polymerase that combines;
B) by its C-end and c) the connection aminoacid sequence that combines;
C) aminoacid sequence of at least one spiral-hair clip-helix domain (HhH) is comprised.
This specification sheets term used " archaeal dna polymerase " relate to can catalytic deoxidation ribonucleoside triphosphote polymerization enzyme.Usually, the primer 3 ' that described enzyme is being hybridized with template DNA sequence holds initial synthesis, and advances to 5 ' end of template DNA chain.
This specification sheets term used " mosaic " relates to the protein that its aminoacid sequence is the fusion product of the aminoacid sequence of at least two kinds of different proteins.Chimeric protein is produced by its expression from the chimeric DNA of encoding chimera aminoacid sequence usually.
This specification sheets term used " archaeal dna polymerase mosaic " or " chimeric dna polymerase " relate to the protein that its aminoacid sequence is the fusion product of the aminoacid sequence of at least two kinds of different proteins (one of them is archaeal dna polymerase), and this protein can the polymerization of catalytic deoxidation ribonucleoside triphosphote.Archaeal dna polymerase mosaic of the present invention comprises N-end region (a) of coding φ 29 type archaeal dna polymerase and C-end region (c) containing at least one HhH structural domain, and (a) combines by being connected aminoacid sequence (b) with (c).
Term used herein " φ 29 type archaeal dna polymerase " relates to containing any archaeal dna polymerase of TPR1 and TPR2 subdomain in its paradigmatic structure territory, and described paradigmatic structure territory provides the ability to pre-polymerization and chain separation coupling for polysaccharase.The example of φ 29 type archaeal dna polymerase used in the present invention is selected from the archaeal dna polymerase be separated from following phage: φ 29, Cp-1, PRD-1, φ 15, φ 21, PZE, PZA, Nf, M2Y, B103, GA-1, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17 or sour Pseudomonas doleiform virus (AcidianusBottle-shapedvirus, ABV).
In the preferred embodiment of this first aspect of the present invention, the amino acid sequence encode φ 29 type archaeal dna polymerase of archaeal dna polymerase of the present invention chimeric (a), described φ 29 type archaeal dna polymerase is selected from the archaeal dna polymerase be separated from following phage: φ 29, Cp-1, PRD-1, φ 15, φ 21, PZE, PZA, Nf, M2Y, B103, GA-1, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17 or sour Pseudomonas doleiform virus (ABV).
Aminoacid sequence and the SEQIDNO:1 of preferred archaeal dna polymerase of the present invention chimeric (a) have at least 80% identity.More preferably the aminoacid sequence of the present invention chimeric (a) and SEQIDNO:1 have at least 90% identity.Even more preferably the aminoacid sequence of the present invention chimeric (a) is SEQIDNO:1.
The exonuclease domain of φ 29 type archaeal dna polymerase is known, and can be modified to reduce exonuclease activity, but retains high processivity and chain separating power.These modified archaeal dna polymerases especially can be used for macromole order-checking.
In the preferred embodiment of this first aspect of the present invention, the amino acid sequence encode of archaeal dna polymerase of the present invention chimeric (a) has the φ 29 type archaeal dna polymerase of modification in exonuclease domain, wherein said modified archaeal dna polymerase preferably has the exonuclease activity being less than 10% compared with corresponding naturally occurring archaeal dna polymerase or " wild-type ", more preferably has the exonuclease activity being less than 1%.In another more preferred, described modified φ 29 type archaeal dna polymerase lacks detectable nucleic acid 5 prime excision enzyme activity relative to corresponding naturally occurring archaeal dna polymerase.
HhH (" spiral-hair clip-spiral ") die body is that the dependent DNA of non-sequence is in conjunction with die body.Structurally, HhH die body is formed by a pair that is connected by hook ring antiparallel alpha-helix.This ring participates in the interaction with DNA, and generally has the consensus sequence be made up of glycine-hydrophobic amino acid-glycine (GhG), and wherein h is hydrophobic amino acid residue, is generally leucine, Isoleucine or α-amino-isovaleric acid.The last glycine of consensus sequence works as the N-terminal residue of second alpha-helix, and hydrophobic residue h contributes to the interaction between two alpha-helixs of die body.
The protein example in its sequence with HhH die body is but is not limited to: the DNA topoisomerase V of Kan Deleshi methane thermophile bacteria, the albumen MutY of intestinal bacteria (Escherichiacoli), Nth, MutM/Fpg, Nei, UvrC, DinP, RecR, UmuC, the albumen RAD1 of DnaE or DnIJ or yeast, RAD2, RAD10, RAD27, RAD55, RAD57, REV1, OGG1, NTG1, NTG2, DIN-7 or EXO-1, and the homologue of these albumen in other biology, other biology described is such as but not limited to subtilis (Bacillussubtilis), Caenorhabditis elegans (Caenorhabditiselegans), hemophilus influenzae (Haemophilusinfluenzae), Methanococcus jannaschii (Methanococcusjannaschii), micrococcus luteus (Micrococcusluteus), hot formic acid methagen (Methanobacteriumthermoformicum) or Salmonella typhimurium (Salmonellatyphimurium).
Therefore, in the preferred embodiment of this first aspect of the present invention, the aminoacid sequence of archaeal dna polymerase of the present invention chimeric (c) comprises at least one the HhH structural domain being selected from following protein:
The topoisomerase V of-Kan Deleshi methane thermophile bacteria;
-colibacillary MutY, Nth, MutM/Fpg, Nei, UvrC, DinP, RecR, UmuC, DnaE or DnIJ;
RAD1, RAD2, RAD10, RAD27, RAD55, RAD57, REV1, OGG1, NTG1, NTG2, DIN-7 or EXO-1 of-yeast; Or
The protein homology thing of the above protein in-subtilis, Caenorhabditis elegans, hemophilus influenzae, Methanococcus jannaschii, micrococcus luteus, hot formic acid methagen or Salmonella typhimurium.
The C-end of the topoisomerase V of Kan Deleshi methane thermophile bacteria is organized as 12 about 50 amino acid whose repetitions, and it is respectively called A-L structural domain, and respectively has two HhH die bodys.
In the preferred embodiment of this first aspect of the present invention, the aminoacid sequence of archaeal dna polymerase of the present invention chimeric (c), comprises at least one the HhH structural domain being derived from Kan Deleshi methane thermophile bacteria topoisomerase V (its aminoacid sequence is SEQIDNO:2).
In the more preferred of this first aspect of the present invention, the aminoacid sequence of archaeal dna polymerase of the present invention chimeric (c) is the SEQIDNO:3 of the sequence in the H structure territory corresponding to Kan Deleshi methane thermophile bacteria topoisomerase V.
In another more preferred of this first aspect of the present invention, the aminoacid sequence of archaeal dna polymerase of the present invention chimeric (c) is the SEQIDNO:3 be combined with SEQIDNO:4 by its C-end, and it corresponds to the pass the sequence in the H structure territory of the Kan Deleshi methane thermophile bacteria topoisomerase V that its C-end is combined with the I structural domain of Kan Deleshi methane thermophile bacteria topoisomerase V.
Term used herein " connection aminoacid sequence " relates to the short amino acid sequence of at least 2 amino acid longs, and it makes it possible to the function of the aminoacid sequence maintaining archaeal dna polymerase of the present invention chimeric (a) and (c).Preferably the connection aminoacid sequence of (b) is made up of 6 amino acid.More preferably the connection aminoacid sequence of (b) is SEQIDNO:5 or SEQIDNO:6.
The present invention relates to the purposes of archaeal dna polymerase mosaic of the present invention for copying template DNA, increasing or check order on the other hand.
The present invention relates to a kind of method for copying template DNA, increasing or check order on the other hand, and described method comprises makes described DNA contact with the reaction mixture comprised at least:
A) archaeal dna polymerase mosaic of the present invention;
B) damping fluid;
C) magnesium chloride;
D) primer; With
E) ribonucleoside triphosphote.
A preferred embodiment of this aspect of the present invention relates to a kind of method for copying template DNA, increasing or check order, described method comprises makes described DNA contact with reaction mixture, described reaction mixture comprises aforesaid ingredients (a)-(e), and comprises polyoxyethylated sorbitan monolaurate, ammonium salt, sylvite or above-mentioned any combination further.
In the present invention in the preferred embodiment of method that copies, increase or check order, the chimeric concentration of archaeal dna polymerase of the present invention is 5nM-75nM.In a more preferred, the chimeric concentration of archaeal dna polymerase of the present invention is 25nM-60nM.In another more preferred, the chimeric concentration of archaeal dna polymerase of the present invention is about 50nM.
In the present invention in the preferred embodiment of method that copies, increase or check order, polyoxyethylated sorbitan monolaurate (
20) concentration is the 0.003%-0.1% of total reaction volume.In a more preferred, the ratio of polyoxyethylated sorbitan monolaurate is the 0.006%-0.05% of total reaction volume.In another more preferred, the ratio of polyoxyethylated sorbitan monolaurate is the 0.01%-0.03% of total reaction volume.In another more preferred, the ratio of polyoxyethylated sorbitan monolaurate is about 0.025% of total reaction volume." total reaction volume " is interpreted as template DNA to join the volume obtained after in reaction mixture.
In the present invention in the preferred embodiment of method that copies, increase or check order, ammonium salt is selected from: ammonium sulfate, ammonium chloride or ammonium acetate.
In the present invention in the preferred embodiment of method that copies, increase or check order, ammonium salt is ammonium sulfate.In a more preferred, the concentration of ammonium sulfate is 30mM-60mM.In another more preferred, the concentration of ammonium sulfate is 40mM-50mM.In another more preferred, the concentration of ammonium sulfate is about 45mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, ammonium salt is ammonium chloride.In a more preferred, the concentration of ammonium chloride is 60mM-120mM.In another more preferred, the concentration of ammonium chloride is 80mM-100mM.In another more preferred, the concentration of ammonium chloride is about 90mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, ammonium salt is ammonium acetate.In a more preferred, the concentration of ammonium acetate is 60mM-120mM.In another more preferred, the concentration of ammonium acetate is 80mM-100mM.In another more preferred, the concentration of ammonium acetate is about 90mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the pH of damping fluid is 7.0-8.5.In a more preferred, the pH of damping fluid is 7.2-8.In another more preferred, the pH of damping fluid is about 7.5.
In the present invention in the preferred embodiment of method that copies, increase or check order, damping fluid is tris-hydrochloric acid, tris-acetic acid or HEPES.In the present invention in the more preferred of method that copies, increase or check order, the pH of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 7.0-8.5.In another more preferred, the pH of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 7.2-8.In another more preferred, the pH of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is about 7.5.
In the present invention in a preferred embodiment in this of the method that copies, increase or check order, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 25mM-50mM.In a more preferred, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 30mM-45mM.In another more preferred, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is about 40mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, sylvite is Repone K or potassium acetate.In a more preferred, the concentration of Repone K or potassium acetate is 30mM-70mM.In another more preferred, the concentration of Repone K or potassium acetate is 40mM-60mM.In another more preferred, the concentration of Repone K or potassium acetate is about 50mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the concentration of magnesium chloride is 2mM-20mM.In a more preferred, the concentration of magnesium chloride is 5mM-15mM.In another more preferred, magnesium chloride is about 10mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the ratio of polyoxyethylated sorbitan monolaurate is the 0.01%-0.03% of cumulative volume, the concentration of ammonium sulfate is 40mM-50mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 30mM-45mM and pH is 7.2-8.0, the concentration of magnesium chloride is 5mM-15mM, and the concentration of Repone K or potassium acetate is 40mM-60mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the concentration of polyoxyethylated sorbitan monolaurate is 0.025% of cumulative volume, the concentration of ammonium sulfate is 45mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 40mM and pH is 7.5, the concentration of magnesium chloride is 10mM, and the concentration of Repone K or potassium acetate is 50mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the ratio of polyoxyethylated sorbitan monolaurate is the 0.01%-0.03% of cumulative volume, the concentration of ammonium chloride is 80mM-100mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 30mM-45mM and pH is 7.2-8.0, the concentration of magnesium chloride is 5mM-15mM, and the concentration of Repone K or potassium acetate is 40mM-60mM.
In the present invention in the more preferred of method that copies, increase or check order, the concentration of polyoxyethylated sorbitan monolaurate is 0.025% of cumulative volume, the concentration of ammonium chloride is 90mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 40mM and pH is 7.5, the concentration of magnesium chloride is 10mM, and the concentration of Repone K or potassium acetate is 50mM.
In the present invention in the preferred embodiment of method that copies, increase or check order, the ratio of polyoxyethylated sorbitan monolaurate is the 0.01%-0.03% of cumulative volume, the concentration of ammonium acetate is 80mM-100mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 30mM-45mM and pH is 7.2-8.0, the concentration of magnesium chloride is 5mM-15mM, and the concentration of Repone K or potassium acetate is 40mM-60mM.
In the present invention in the more preferred of method that copies, increase or check order, the concentration of polyoxyethylated sorbitan monolaurate is 0.025% of cumulative volume, the concentration of ammonium acetate is 90mM, the concentration of damping fluid tris-hydrochloric acid, tris-acetic acid or HEPES is 40mM and pH is 7.5, the concentration of magnesium chloride is 10mM, and the concentration of Repone K or potassium acetate is 50mM.
This specification sheets term used " copies " and relates to from template DNA synthesis complementary DNA.
This specification sheets term used " amplification " relates to the copy number increasing template DNA.
This specification sheets term used " order-checking " relates to the order measuring template DNA Nucleotide.
" contact " is interpreted as such fact, namely under primer extension conditions, hatches template DNA and reaction mixture.
Term used herein " primer " to relate to when it is under primer extension conditions and can synthesize as DNA the oligonucleotide that starting point works.Preferred primer is deoxyribose oligonucleotide.
Prepare primer by any suitable method, described method comprises such as but not limited to direct chemosynthesis.Can be hybridize design of primers (Auele Specific Primer) with the specific deoxynucleotide sequence of template DNA, or can random synthesis (random primer).
This specification sheets term used " Auele Specific Primer " relates to the primer of the specific deoxynucleotide sequence complementation of its sequence and template DNA to be amplified.
" complementation " is interpreted as such fact, namely primer can with the area hybridization of template DNA, make to can be used as when it is under primer extension conditions DNA and synthesize starting point and work.Preferably the region of this region and template DNA has 100% complementarity.In other words, hydrogen bond can be formed with the Nucleotide that exists in single-stranded template with each Nucleotide in the region of Primers complementary.But the personnel with this area general knowledge will admit, can play a role to implement the method for the present invention for copying, increasing or check order containing the primer relative to template DNA with the region being less than 100% complementarity.
Term " random primer " relates to its sequence random synthesis and primer for synthesizing at the random site initiate dna of template DNA.Usually, in the present invention in the method that copies, increase or check order, one group of random primer is used.Term " random primer " relates to and has stochastic sequence and a series of primers for synthesizing at the random site initiate dna of template DNA.
In the present invention in the preferred embodiment of method that copies, increase or check order, primer is specific.
In the present invention in another preferred embodiment of method of copying, increase or checking order, primer is random.Preferably arbitrarily primer is subject to the effect being protected from 3 '-5 ' exonuclease.And more preferably random primer is oligonucleotide, " Hexanucleotide " or " sexamer " of 6 Nucleotide, it is subject to the effect being protected from 3 '-5 ' exonuclease.
The statement used of this specification sheets " is subject to the effect being protected from exonuclease " and relates to modified primer, makes the nucleolysis of any 3 '-5 ' exonuclease activity existed in its opposing archaeal dna polymerase mosaic of the present invention.
In the present invention in the method that copies, increase or check order, more than a kind of primer can be used, Auele Specific Primer and/or random primer can be used.
In the present invention in the preferred embodiment of method that copies, increase or check order, the concentration of primer is 2 μMs-100 μMs.In a more preferred, the concentration that primer is 20 μMs-80 μMs.In another more preferred, the concentration of primer is 40 μMs-60 μMs.In another more preferred, the concentration of primer is about 50 μMs.
This specification sheets term used " ribonucleoside triphosphote " relates to the organic molecule formed by the covalent linkage of pentose, nitrogen base and three phosphate groups.
Term ribonucleoside triphosphote comprises deoxynucleoside triphosphate (dNTP), such as but not limited to dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof.Preferred deoxynucleoside triphosphate is dATP, dTTP, dGTP and dCTP.Even more preferably these four kinds of dNTP such as to be at the molar conditions.In the preferred embodiment of the present invention in this, the concentration of deoxynucleoside triphosphate is 100 μMs-800 μMs.In a more preferred, the concentration of deoxynucleoside triphosphate is 200 μMs-600 μMs.In another more preferred, the concentration of deoxynucleoside triphosphate is about 500 μMs.
Term ribonucleoside triphosphote also comprises dideoxyribonucleoside triphosphate (ddNTP), such as but not limited to ddATP, ddCTP, ddITP, ddUTP, ddGTP, ddTTP or derivatives thereof.
In the present invention in the certain preferred embodiments of method that copies, increase or check order, by the technical mark at least one ribonucleoside triphosphote known in the present situation of this area or a kind of primer.The Nucleotide be labeled can be such as deoxynucleoside triphosphate or dideoxyribonucleoside triphosphate.Detectable mark comprises such as radio isotope, fluorescent mark, chemiluminescent labeling, bioluminescence marker or enzyme labelling.
This specification sheets term used " template DNA " relates to the DNA molecular of the substrate that can be used as the synthesis of complementary dna chain; Namely it relates to DNA molecular that is to be copied, that increase or check order.In a preferred embodiment, template DNA is plasmid DNA.In another preferred embodiment of the present, template DNA is genomic dna.
Copying, increase or checking order of template DNA is implemented under primer extension conditions.Statement " primer extension conditions " refers to the condition of the synthesis that wherein can occur in dependence template DNA initial in primer.
According to the method for the present invention for copying, increasing or check order, by Thermal Cycling or template DNA synthesis occurs under constant temperature substantially.
" isothermal condition " is interpreted as constant temperature substantially.According to the method for the present invention for copying, increasing or check order, preferred template DNA synthesis occurs under constant temperature substantially.More preferably constant temperature is 25-40 DEG C substantially, is even more preferably about 30 DEG C.
Allow the large metering method of DNA cloning well-known in the present situation of this area.Certain methods needs Thermal Cycling, such as but not limited to polymerase chain reaction (PCR).Other method does not need Thermal Cycling, but carry out under constant temperature substantially, such as but not limited to rolling loop type amplification (RCA), multiple displacement amplification (multipledetachmentamplification, MDA), strand displacement amplification (SDA) or ring mediated amplification (LAMP).According to the inventive method by Thermal Cycling or template DNA amplification occurs under constant temperature substantially.
According to amplification method of the present invention, preferably by rolling loop type amplification (RCA), the amplification being carried out template DNA by multiple displacement amplification (MDA), strand displacement amplification (SDA) or ring mediated amplification (LAMPA).
The present invention relates on the other hand a kind of comprising and is suitable for implementing the present invention for the test kit of the integral part of method that copies, increase or check order or device.
The present invention relates on the other hand a kind of for implementing the test kit of method of the present invention for copying, increasing or check order, and described test kit comprises:
A) archaeal dna polymerase mosaic of the present invention;
B) damping fluid; With
C) magnesium chloride.
In the present invention in this in preferred embodiment, test kit also comprises polyoxyethylated sorbitan monolaurate, ammonium salt, sylvite or above-mentioned any combination.
Preferred described ammonium salt is selected from: ammonium sulfate, ammonium chloride or ammonium acetate.
Preferred described sylvite is Repone K or potassium acetate.
In the present invention in this in preferred embodiment, test kit also comprises primer.In a more preferred, primer is the random primer being subject to being protected from the effect of 3 '-5 ' exonuclease.
In the present invention in this in preferred embodiment, test kit also comprises ribonucleoside triphosphote.Such as, in the present invention in this in more preferred, test kit also comprises deoxynucleoside triphosphate and/or dideoxyribonucleoside triphosphate.
In the present invention in this in preferred embodiment, test kit comprises at least one nucleoside triphosphate or a kind of labeled primer.The nucleosides of mark can be such as deoxynucleoside triphosphate or dideoxyribonucleoside triphosphate.
Test kit also can include but not limited to any type of damping fluid, antipollution reagent etc.On the other hand, test kit can comprise for put into practice and be used for its optimize needed for all upholders and container.Preferred reagent box also comprises the specification sheets for implementing the inventive method.
The term running through this specification sheets and claims " comprises " and variant does not get rid of other technical characteristic, additive, component or step.To those skilled in the art, can partly from this specification sheets and partly know other object, advantage and feature by inference from enforcement of the present invention.Illustrate by way of example and provide the following drawings and embodiment, it does not limit the present invention.
Accompanying drawing explanation
Fig. 1 shows
20 and (NH4)
2sO
4effect in φ 29DNA polymeric enzymatic amplification ability.Implement to measure as described in main text under the plasmid DNA (4.2kpb) of amount shown exists.After hatching 5 hours at 30 DEG C, analytical reaction thing as described in main text.The left side is the linear DNA fragment obtained after digesting φ 29DNA with HindIII being used as DNA length mark.
Fig. 2 shows φ 29DNA polysaccharase and exists
20 and (NH4)
2sO
4to the amplification of the plasmid DNA (flying grams magnitude) of difference amount under existence.0.025% as described in main text
20 and 45mM (NH4)
2sO
4implement under existence to measure.DNA length mark is used identical with Fig. 1.
Fig. 3 shows NH
4 +the effect of ion in φ 29DNA polymeric enzymatic amplification ability.0.025% as described in main text
20 and shown ammonium salt and amount shown plasmid DNA (4.2kpb) exist under implement measure.After hatching 6 hours at 30 DEG C, analytical reaction thing as described in main text.DNA length mark is used identical with Fig. 1.
Fig. 4 shows φ 29DNA polysaccharase and exists
20 and (NH4)
2sO
4to the amplification of difference amount Bacillus subtilis genes group DNA under existence.0.025% as described in main text
20 and 45mM (NH4)
2sO
4implement under existence to measure.DNA length mark is used identical with Fig. 1.
Fig. 5 display is by by 0.025%
20 and 45mM (NH
4)
2sO
4join for based on
remarkable improvement represented in the current reaction buffer of the commercial reagents box (the Illustra test kit of GeneralElectricsHealthCare) of the DNA cloning of archaeal dna polymerase.Implement to measure as described in main text.DNA length mark is used identical with Fig. 1.
Fig. 6 show build mosaic HAY, HGT, HIAY and HIGT in accordance with the figure of different step.
Fig. 7 shows the retardance of primer/template DNA molecule in gel caused by natural and chimeric φ 29DNA polysaccharase.Make the hybrid molecule (dsDNA) of 15 base/21 bases marked 5 ' with natural φ 29DNA polysaccharase or hatch under condition described herein with shown chimeric dna polymerase.The migration of free dsDNA and polymerase-DNA complexes is detected by its radioautograph after electrophoretic separation in 4% (w/v) native polyacrylamide gel (monomer of 80: 1: bisacrylamide).
Fig. 8 shows the DNA that natural and chimeric φ 29DNA polysaccharase causes to be copied forward.(A) what natural and chimeric φ 29DNA polysaccharase caused copies with the M13DNA of chain separation coupling.As described hereinly carry out copying of the M13DNA of 250ng with single primer with natural or chimeric φ 29DNA polysaccharase (30nM).The position display of the M13DNA of unit length on the right.(B) synthesis forward caused by natural and chimeric φ 29DNA polysaccharase.Implement to measure by shown decrescence archaeal dna polymerase concentration under the condition identical with (A).After hatching 20 minutes at 30 DEG C, processing sample as described in (A).
Fig. 9 shows the rolling loop type amplification of the multiple initiation of the 10fg plasmid DNA that natural and chimeric φ 29DNA polysaccharase causes.Implement to measure as described in main text under shown buffer B and 50nMDNA polysaccharase exist.DNA length mark is used identical with Fig. 1.
Figure 10 shows the whole genome amplification of the multiple initiation of the 100fg Bacillus subtilis genes group DNA that natural and chimeric φ 29DNA polysaccharase causes.As described herein enforcement under shown buffer B and 50nMDNA polysaccharase exist measures.DNA length mark is used identical with Fig. 1.
Embodiment
The following specific embodiment provided in this patent document plays a part to illustrate characteristic of the present invention.Comprising these embodiments only in order to illustrate object, the restriction of the present invention to advocating need not being interpreted as herein.Therefore, following embodiment is illustrated the present invention and is not limited its Application Areas.
embodiment 1 is for the optimization of the experiment condition of the multiple initiation DNA amplification of φ 29DNA polysaccharase
Already showed φ 29DNA polysaccharase and increased 10 from a few pik cyclic DNA
4-10
6doubly.For this purpose, use containing 40mMtris-HCl (pH7.5), 50mMKCl and 10mMMgCl
2the reaction buffer of (hereinafter referred to as buffer A).Test different stain remover and salt condition on the impact of the DNA cloning ability of φ 29DNA polysaccharase after, find 0.025%
20 and 45mM (NH
4)
2sO
4join in buffer A simultaneously, the amplification of the limited amount DNA provided is provided greatly.
For the reaction conditions of amplified plasmid dna.-mixtures incubated contains 12.5 μ l buffer A, 50 μMs of protected sexamers from the effect of 3 '-5 ' exonuclease, each deoxynucleoside triphosphate (dCTP, dGTP, dTTP and dATP) of 500 μMs, plasmid DNA (size is 4.2kbp) of amount shown, and adds (NH by shown
4)
2sO
445mM or 0.025%
20 or the two combination.By hatching 3 minutes at 95 DEG C and cool 5 minutes subsequently in ice, make DNA sex change.Add initial action after 50nM φ 29DNA polysaccharase, by be heated to 65 DEG C continue to hatch at 30 DEG C for 10 minutes after termination reaction.In order to analytical results, from reactant, take out 1 μ l sample, with the DNA of EcoRI restriction endonuclease digest amplification, and in 0.7% sepharose electrophoresis.By detecting DNA with ethidium bromide to gel-colored.
For the reaction conditions of amplifying genom DNA.-mixtures incubated contains 12.5 μ l buffer A, 45mM (NH
4)
2sO
4, 0.025%
20, the Bacillus subtilis genes group DNA (size is 4Mpb) of 50 μMs of protected sexamers from the effect of 3 '-5 ' exonuclease, 500 μMs of each deoxynucleoside triphosphates (dCTP, dGTP, dTTP and dATP) and amount shown.Within 5 minutes, DNA sex change is made by hatching 3 minutes at 95 DEG C and cool in ice subsequently.Add initial action after 50nM φ 29DNA polysaccharase, by be heated to 65 DEG C continue to hatch at 30 DEG C for 10 minutes after termination reaction.In order to analytical results, from reactant, take out 1 μ l sample, and in 0.7% sepharose electrophoresis.By detecting DNA with ethidium bromide to gel-colored.
Fig. 1 display adds 45mM (NH
4)
2sO
4with 0.025%
20 in the effect in a small amount of plasmid DNA of providing of increasing.As shown, as the DNA using 100fg to provide, φ 29DNA polysaccharase standard buffer solution A does not produce detectable any amplified production.In these reaction conditionss, add 0.025% when there is not DNA
20 cause occurring trace amount DNA product, and major part may be the non-specific DNA cloning because hybridization and the extension of random sexamer primer causes.Same trace is observed with the DNA that 10fg provides.But, under the DNA existence that 100fg provides, add 0.025%
20 make φ 29DNA polysaccharase to produce can the plasmid of amplification of detection limit.The ultimate production of the DNA of specificity or non-specific amplification shows, by 0.025%
20 join in buffer A the amplification ability facilitating φ 29DNA polysaccharase.Similar effect is observed with NP40 stain remover.On the contrary, other stain remover such as TritonX100 and TritonX114 analyzed does not promote the amplification ability (not shown) of φ 29DNA polysaccharase.By 0.025%
20 and 45mM (NH
4)
2sO
4joining in buffer A simultaneously, there are two kinds of results in the output and specificity of amplified production: 1) can't detect DNA cloning when there is not provided DNA; 2) even if when provided DNA amount is low to moderate 10fg, also obtained the plasmid DNA of the unit length of some μ g by amplification.In contrast, by 45mM (NH
4)
2sO
4join in buffer A and any improvement is not produced to φ 29DNA polymeric enzymatic amplification ability.
Therefore, the conclusion that can draw is, by 0.025%
20 and 45mM (NH
4)
2sO
4join (hereinafter referred to as buffer B) in buffer A to produce obviously optimize for implementing the multiple experiment condition causing amplification of φ 29DNA polysaccharase to cyclic DNA simultaneously, the two be all the DNA of the finite quantity (10fg) that amplification provides definitely required reagent.In fact, as seen in Figure 2, buffer B is used to make φ 29DNA polysaccharase can synthesize micrograms of DNA after 6 hours of reaction by the plasmid amount provided being low to moderate 0.1fg (about 24 molecules).As quality control, create the linear dsDNA fragment of 4.2kb with EcoRI digest amplification product, this shows that amplified production is the tandem sequence repeats of original plasmid really.In addition, buffer B also prevents non-specific DNA cloning (swimming lane see corresponding to the reaction not providing DNA to implement in Fig. 2).
Fig. 3 shows ammonium ion and 0.025%
20 are improving the effect in a small amount of plasmid DNA of amplification.0.025%
20 and shown ammonium salt exist under in previous described condition implement measure.As Fig. 3 is observable, NH
4cl and NH
4cH
3both COO have and (NH in the output of amplified production and specificity
4)
2sO
4similar effect.This result shows, aforementioned (NH
4)
2sO
4effect in amplification finite quantity plasmid DNA is due to NH
4 +ion.
Whether being also applicable to amplifying genom DNA to measure above-mentioned optimal conditions, under the Bacillus subtilis genes group DNA (long 4Mpb) of Finite Concentration exists, having carried out the mensuration of the same-type implemented.As shown in Figure 4, in buffer B, 0.025% is had
20 and 45mM (NH
4)
2sO
4prevent non-specific DNA cloning (swimming lane without the DNA provided) on the one hand, make φ 29DNA polysaccharase can produce detectable and specific genomic DNA amplification on the other hand, even if when the DNA using 10fg to provide, namely compare the amount that current business genome amplification test kit recommends and be reduced to 1/10
6.
Add 0.025% to measure simultaneously
20 and 45mM (NH
4)
2sO
4whether raising be used for based on
the amplification efficiency of the current commercial reagents box of the DNA cloning of archaeal dna polymerase, the plasmid DNA amplification carried out as Fig. 1,2 types identical with as described in 3 measures.Fig. 5 display is by by 0.025%
20 and 45mM (NH
4)
2sO
4join remarkable improvement represented in the current reaction buffer of Illustra test kit (GEHealthCare).As observable, according to the recommendation of supplier, with Illustra test kit only when provided plasmid amount is equal to or greater than 10pg, just can increase in mode detectable in sepharose.In contrast, by 0.025%
20 and 45mM (NH
4)
2sO
4join in the reaction buffer of Illustra test kit, the amount of the DNA increased needed for significantly reducing, can be observed amplified production from provided 1fg plasmid DNA, this relates to the raising of four orders of magnitude of amplification simultaneously.
embodiment 2. improves the amplification ability of φ 29DNA polysaccharase by adding HhH structural domain
Make φ 29DNA polysaccharase and one or two HhH domain fusion, build the new archaeal dna polymerase of the DNA binding ability that tool improves, it makes it possible to use the DNA comparatively in a small amount provided.The structure inspection of φ 29DNA polysaccharase causes the C-terminal fusion selecting HhH structural domain and this enzyme, because this end (end of subdomain thumb) is near the feeder connection of top dsDNA sequence.The N-terminal fusion of HhH structural domain and φ 29DNA polysaccharase, by damaging inherent chain separating power, occurs near aminoterminal because biological chemistry and structured data show that female DNA untwists.In addition, to hybridize the dsDNA combination partly formed be inappropriate for improving sexamer primer and template DNA HhH structural domain to be positioned at N-end.In this case, for amplification, there is destruction at φ 29DNA polysaccharase N-terminal fusion sequence (His) 6.
2.1 chimeric dna polymerase constructs
In order to prepare mosaic HIAY and HIGT, entrust GenScriptCorporation synthetic DNA fragment, described fragment contains the HhH structural domain H (56 amino acid) of coding Kan Deleshi methane thermophile bacteria topoisomerase V and Nucleotide (the GenBank code AF311944 of I (51 amino acid), Pavlov etc., ProcNatlAcadSciUSA.2002; 99:13510-13515), between the EcoRV site this DNA fragmentation being cloned into commercial vector pUC57.The plasmid pUC57-HhH obtained is used as the DNA fragmentation that template is used for by pcr amplification encoding domain H and I.Therefore, primer 3 (SEQIDNO:7) produces DNAI and the II fragment of 369bp respectively together with primer 1 (SEQIDNO:8) or 2 (SEQIDNO:9).Except the KasI site of being introduced by two primers, primer 1 also introduces sequence (Pavlov etc., the ProcNatlAcadSciUSA.2002 of coding SEQIDNO:5 joint; 99:13510-13515), and the nucleotide sequence of primer 2 introducing coding SEQIDNO:6 joint (previously at Sun etc., Proteins.2006; SEQIDNO:10 linker derivative described in 65:231-238).Primer 3 sequence (see chimeric dna polymerase construct sketch in Fig. 6) containing coding 6 histidine residues then terminator codon and BamHI site.
Meanwhile, by pJLw2 plasmid derivative thing (L á zaro etc., the MethodsEnzymol.1995 of the gene containing coding φ 29DNA polysaccharase (572 amino acid); 262:42-49) as template, for with the PCR reaction comprising the primer 4 (SEQIDNO:11) in 5 ' HindIII site and primer 5 (SEQIDNO:12) or 6 (SEQIDNO:13) and implement, to obtain fragment III and the IV of 1757bp respectively.Therefore, fragment III and IV should, containing coding φ 29DNA polysaccharase, then be the DNA of SEQIDNO:5 (fragment III) and SEQIDNO:6 (fragment IV) sequence also comprising KasI site.Purified fragments I-IV in 0.7% sepharose, then digests with KasI.Connect DNA fragmentation I and III and II and IV of digestion with T4DNA ligase enzyme, obtain the linear DNA of the 2108bp of encoding chimera body HIAY (fragment V) and HIGT (fragment VI) respectively.The product that purifying connects in 0.7% sepharose, then uses BamHI and HindIII endonuclease digestion.By the product of the digestion of Purified in electrophoresis in sepharose.Fragment V and VI is cloned into (Tabor etc., ProcNatlAcadSciUSA.1985 in carrier pT7-4 the most at last; 82:1074-1078).By mosaic HIAY (structural domain H and I of φ 29DNA polysaccharase+SEQIDNO:5 joint+topoisomerase V) and HIGT (structural domain H and I of φ 29DNA polysaccharase+SEQIDNO:6 joint+topoisomerase V) as template, to pass through
(Stratagene) directed Mutagenesis Kit builds mosaic HGT and HAY after terminator codon being inserted the H fragment of topoisomerase V respectively.By carrying out DNA sequence dna to full genome order-checking and there is not the confirmation of sudden change in addition.Accommodating e. coli bl21 (DE3) the cells chimeric dna polymerase of the mosaic gene be cloned in pJLw2 plasmid derivative thing, then substantially as (L á zaro etc., MethodsEnzymol.1995; 262:42-49) described purifying.
In a word, the chimeric dna polymerase of acquisition is as follows:
HAY: φ 29DNA polysaccharase-SEQIDNO:5-HhHH (635aa; About 73kDa)
HGT: φ 29DNA polysaccharase-SEQIDNO:6-HhHH (635aa; About 73kDa))
HIAY: φ 29DNA polysaccharase-SEQIDNO:5-HhHH-I (692aa; About 80kDa)
HIGT: φ 29DNA polysaccharase-SEQIDNO:6-HhHH-I (692aa; About 80kDa)
2.2. the DNA binding ability of chimeric dna polymerase.
In order to the HhH die body being determined at φ 29DNA polysaccharase end merge whether give that mosaic improves with the binding ability of DNA, in gel, carried out the electrophoretic migration retardation assays of DNA.
Condition determination.-provide the oligonucleotide of 15 bases (SEQIDNO:14) by Isogen and also there is the oligonucleotide of 21 bases (SEQIDNO:15) that 5 ' of 6 Nucleotide extend except the sequence of the oligonucleotide complementation with 15 bases.5 ' with [γ-
32p] oligonucleotide of ATP and T4 kinases polynucleotide labelling 15 bases.Oligonucleotide hybridization that is lower and 21 bases is there is at 0.2MNaCl and 60mM (pH7.5) tris-HCl at the oligonucleotide of 5 ' 15 bases marked.With the oligonucleotide analysis of oligonucleotide molecules/21 base in 5 ' 15 bases marked and the interaction of natural or chimeric φ 29DNA polysaccharase of hybridization.The mixtures incubated of 20 μ l final volume contains 50mM (pH7.5) tris-HCl, 1mM dithiothreitol (DTT), 10mMMgCl
2, 20mM ammonium sulfate, 0.1mg/ml bovine serum albumin (BSA), 4% glycerine, the oligonucleotide molecules of 1nM15 base/21 base and the natural of shown concentration or chimeric φ 29DNA polysaccharase.After hatching 5 minutes at 4 DEG C, make sample electrophoresis in 4% polyacrylamide gel (w/v) (monomer: binary 80: 1) containing 12mM (pH7.5) tris-acetic acid and 1mMEDTA, develop the color with 8V/cm in the same buffer of 4 DEG C, it is substantially as (Carthew etc., Cell.1985; 43:439-448).After radioautograph, polysaccharase-dsDNA mixture is detected as the transport (retardance) in the DNA migration position of mark.
In these conditions, the natural φ 29DNA polysaccharase hybridization oligonucleotide acid molecule of the mark of 15 base/21 bases produces single retardance band (see Fig. 7), the oligonucleotide molecules of described hybridization can be interpreted as stabilized enzyme-DNA mixture (M é ndez etc., the JBiolChem.1994 that can be used for being polymerized; 269:30030-30038).The DNA binding ability that mosaic HAY, HGT and HIGT display is stronger than natural enzyme, because most of substrate separates when 9.5nM concentration, does not resemble the concentration that native DNA polymerase needs about 2 times.Mosaic HIAY has similar to natural polymerization enzyme or even lower DNA binding ability.Can draw from these results, generally speaking, the C-end HhHH and the H+I structural domain of topoisomerase V being added to φ 29DNA polysaccharase gives the DNA binding ability improved, but has exception, such as, when mosaic HIAY.
2.3. by rolling cycle replication that chimeric dna polymerase carries out.
The DNA whether improvement that the DNA obtained to measure C-end by the HhH structural domain of topoisomerase V being added to φ 29DNA polysaccharase combines affect polymerization activity and impact and chain separation coupling on the other hand on the one hand synthesizes forward, implement with M13 primer and copy mensuration, wherein archaeal dna polymerase is polymerized from 3 '-OH group of DNA oligonucleotide.In this mensuration, first replication cycle does not need chain to be separated, but once complete, polysaccharase just finds 5 ' of primer to hold, and needs thus to be initiatively separated to continue later replication cycle (rolling loop type type).
Condition determination.-M13mp18ssDNA is existed at 0.2MNaCl and 60mM (pH7.5) tris-HCl lower to hybridize with universal primer, by the molecule that obtains as primer/template, that analyzes that chimeric dna polymerase carries out is polymerized with the DNA forward of chain separation coupling.25 μ l mixtures incubated contain 50mM (pH7.5) tris-HCl, 10mMMgCl
2, 1mM dithiothreitol (DTT), 4% glycerine, 0.1mg/mlBSA, 40 μMs of dCTP, dGTP, dTTP and [α-
32p] dATP (1 μ Ci), 250ng sequence SEQIDNO:16 the natural or chimeric φ 29DNA polysaccharase of M13mp18ssDNA and 30nM that causes of oligonucleotide.After hatching the shown time at 30 DEG C, carry out termination reaction by adding 0.1%EDTA, 10mM-SDS, by SephadexG-50 post filtered sample.Relative reactivity is calculated from the Cerenkov radiation corresponding to excluded volume.For Analyzing on Size, by making the DNA sex change of mark with 0.7MNaOH process, in 0.7% alkaline agarose gels, carry out electrophoresis, it is as (McDonell etc., JMolBiol.1977; 110:119-146).After electrophoresis, by detecting the M13mp8ssDNA of unit length with ethidium bromide staining, then desiccant gel radioautograph.
As shown in Figure 8 A, the chain separating power of chimeric dna polymerase is not disturbed in the existence of the great HhH of being found to be structural domain.The amount of the DNA of mosaic HAY, HGT, HIAY and HIGT synthesis is greater than the amount (being respectively 4,5,5 and 7 times) of natural φ 29DNA polysaccharase synthesis.Reproduction speed similar to the speed obtained with natural φ 29DNA polysaccharase (under mosaic HAY and HGT situation) or even faster (mosaic HIGT and HIAY).This result shows, in mosaic, φ 29DNA polysaccharase C-end exists HhH structural domain, improves the utilization of the template DNA during rolling cycle replication.
2.4. by chimeric dna polymerase carry out to pre-polymerization.
φ 29DNA polysaccharase is the example copied forward for DNA, because it can mix the base that is greater than 70kb and not dissociate with DNA when there is not accessory protein.Therefore, analyze φ 29DNA polysaccharase C-terminal fusion HhH structural domain in chimeric dna polymerase and whether affect the processivity of polymerization.
Condition determination.-use the enzyme/DNA analysis of the different ratios processivity of chimeric dna polymerase.The mixtures incubated of 25 μ l contains 50mM (pH7.5) tris-HCL, 10mMMgCl
2, 1mM dithiothreitol (DTT), 4% glycerine, 0.1mg/mlBSA, 40 μMs of dCTP, dGTP, dTTP and [α-
32p] M13mp18ssDNA that causes of dATP (1 μ Ci), 250ng and the shown natural φ 29DNA polysaccharase decrescence measured or chimeric dna polymerase.After hatching 20 minutes at 30 DEG C, carry out termination reaction by adding 10mMEDTA-0.1%SDS, by SephadexG-50 post filtered sample.For Analyzing on Size, by making the DNA sex change of mark with 0.7MNaOH process, and in 0.7% alkaline agarose gels electrophoresis.The processivity of polymerization is assessed by the length copying product by the archaeal dna polymerase/DNA analysis of decrescence ratio.
As shown in Figure 8 B, according to DNA polymerization model forward, decrescence enzyme/the DNA of ratio does not change the length of the extension products of natural or chimeric φ 29DNA polysaccharase synthesis.
the multiple initiation of the plasmid DNA of 2.5. being undertaken by natural and chimeric φ 29DNA polysaccharase
rolling loop type amplification (RCA).
As mentioned above, the high processivity that φ 29DNA polysaccharase has and chain separating power, be AmershamBiosciences/MolecularStaging exploitation for the basis of more one of the effective ways of the isothermal ssDNA that increases, wherein φ 29DNA polysaccharase and random sexamer primer be combined through pik cyclic plasmid [Templiphi
tM(www.gehealthcare.com) chain] is separated and realizes 10
4-10
6isothermal doubly accurately increases.Contain at its C-end (His) of fusion
6the result display of the φ 29DNA polysaccharase of sequence, although it is fine by single primer efficiency during rolling cycle replication, can not produce detectable amplified production during the RCA of multiple initiation.Other mutant derivatives for φ 29DNA polysaccharase is so equally, and other mutant derivatives described shows larger avidity for dNTP, and it copies M13DNA with the level of native DNA polymerase, but can not obtain amplified production.Therefore, although the fusion of HhH structural domain and φ 29DNA polysaccharase C-end improves natural enzyme carries out rolling cycle replication endogenous capacity with single primer, expectability does not have similar efficiency to improve during the RCA of multiple initiation.
Condition determination.Mixtures incubated in-12.5 μ l buffer B as provide containing 10fg plasmid DNA (4.2kpb).In order to make provided DNA sex change, sample being hatched 3 minutes at 95 DEG C, then in ice, cooling 5 minutes.Initial action is carried out by adding the natural or chimeric φ 29DNA polysaccharase of 50nM.After hatching the shown time at 30 DEG C, within 10 minutes, carry out termination reaction by being hatched at 65 DEG C by sample.Digest often kind of reactant of 1 μ l with EcoRI, then analyzed by electrophoresis in 0.7% sepharose.By detecting the DNA of amplification with ethidium bromide staining after electrophoresis.
As shown in Figure 9, natural φ 29DNA polysaccharase produced detectable amplified production from reaction 4 hours.Mosaic HAY produced detectable amplified production from 4 hours, and after 5 hours, the gross product amount of amplification is 2 times that obtain with natural φ 29DNA polysaccharase.Mosaic HGT produce can with the DNA of the amplification of a great deal of obtained with mosaic HAY; Interestingly within the shorter reaction time (3 hours), maximum amplification output is obtained.The maximum of the DNA increased with mosaic HIAY with HIGT to obtain with natural φ 29DNA polysaccharase similar, but in mosaic HGT situation, described maximum production autoreaction is tracked down and recovered for 3 hours.After the DNA digesting 4 kinds of mosaic synthesis with EcoRI, the DNA of the amplification more than 80% produces the dsDNA fragment of 4.2kb, and this shows that most of amplified production is made up of the tandem sequence repeats of original plasmid really.
This result shows, chimeric dna polymerase is stronger than the ability of the plasmid DNA of natural φ 29DNA polymeric enzymatic amplification finite quantity (10fg).
the multiple initiation of 2.6 genomic dnas undertaken by natural and chimeric φ 29DNA polysaccharase
dNA cloning.
Except the RCA technology of above-mentioned multiple initiation, based on φ 29DNA polysaccharase character and the random sexamer primers in combination of use, develop method (Dean etc., the ProcNatlAcadSciUSA.2002 for the full-length genome that increases that are called multiple displacement amplification (MDA); 99:5261-5266).Based on the Genomiphi of the method
tM(GEHealthcare) and
(Qiagen) test kit needs minimum is the genomic dna of 10ng.Whether to improve the ability of amplification finite quantity genomic dna in order to study chimeric dna polymerase relative to natural φ 29DNA polysaccharase, having carried out the MDA of Bacillus subtilis genes group DNA.
Condition determination.Mixtures incubated in-12.5 μ l buffer B contains 100fg Bacillus subtilis genes group DNA.In order to make DNA sex change, sample being hatched 3 minutes at 95 DEG C, then in ice, cooling 5 minutes.Initial action is carried out by adding the natural or chimeric φ 29DNA polysaccharase of 50nM.After hatching the shown time at 30 DEG C, within 10 minutes, carry out termination reaction by being hatched at 65 DEG C by sample.Often kind of reactant of 1 μ l is analyzed by electrophoresis in 0.7% sepharose.By detecting the DNA of amplification with ethidium bromide staining after electrophoresis.
As shown in Figure 10, in above-mentioned experiment condition, natural φ 29DNA polysaccharase produces detectable amplified production after hatching 5 hours.Mosaic HAY also produces amplified production at this moment, but the STb gene amount of amplification after 6 hours is more much more than the amount obtained with natural φ 29DNA polysaccharase.Also can be observed, all the other mosaics HGT, HIAY and HIGT produce obvious amplified production in shorter minute (3 hours), total amplification amount now and similar (HIAY and HIGT) or much more (HGT) that observe after 6 hours with natural φ 29DNA polysaccharase.These results show, HhH structural domain exist for the ability that chimeric dna polymerase provides the amplifying genom DNA improved relative to natural φ 29DNA polysaccharase.
the measurement of 2.7 chimeric dna polymerase accuracy
Mixture (2 μ lNewEnglandBiolabs (NEB) 10XEcoRI damping fluids, 0.5 μ l [10 units] NEBEcoRI endonuclease and 14.5 μ lH are limited at 17 μ l
2o), under existing, hatch and often plant sample from the 3 μ l tested shown in Fig. 9, to obtain the linear monomer of amplification plasmid corresponding to multiple rolling loop type amplification 10fg plasmid DNA.After hatching 1 hour at 37 DEG C, by Qiagen gel-extraction test kit column purification DNA, and at the middle wash-out of 30 μ lTE damping fluids (10mM (pH7.5) tris-HCl, 1mMEDTA).By by itself and 2 μ lNEB10X ligase enzyme damping fluids, 8 μ lH
2o and 0.5 μ l (200 units) NEBT4DNA ligase enzyme is hatched, and again connects often kind of elutriant of 10 μ l.At 16 DEG C, reactant is hatched a night, often plant reactant transformation of E. coli XL-1Blue competent cell with 2 μ l.Obtain about 1000 strain transformant with often kind of amplification sample, and do not obtain transformant with containing with the control sample of the 10fg4.2kpb plasmid of the mode process similar to above-mentioned each sample.From each conversion, select two clones, check order according to the corresponding plasmid of standard method purifying.Oligonucleotide for checking order is: pT7-N (SEQIDNO:17), sp4+10 (SEQIDNO:18) and sp10+7 (SEQIDNO:19).Add up to and 4918 of the often kind of plasmid increased by natural and chimeric dna polymerase non-overlapped Nucleotide are checked order.Result is shown in Table 1, and it shows the synthesis accuracy that chimeric dna polymerase is similar to natural enzyme.
The polymerization accuracy of the natural and chimeric φ 29DNA polysaccharase of table 1.
| Polysaccharase | Sudden change |
| Natural | 0 |
| HAY | 0 |
| HGT | 0 |
| HIAY | 1 (G is converted to T) |
| HIGT | 0 |
As described in main text, 4918 non-overlapped Nucleotide of the often kind of plasmid increased by natural and chimeric dna polymerase are checked order.
Claims (39)
1. an archaeal dna polymerase mosaic, described mosaic is made up of following:
A) by its C-end and b) the Φ 29 type archaeal dna polymerase be made up of aminoacid sequence SEQIDNO:1 that combines;
B) by its C-end and c) the connection aminoacid sequence that combines, wherein said connection aminoacid sequence is SEQIDNO:5 or SEQIDNO:6;
C) aminoacid sequence containing at least one spiral-hair clip-spiral (HhH) structural domain, wherein the aminoacid sequence c) SEQIDNO:3 that is SEQIDNO:3 or is combined with SEQIDNO:4 by its C-end.
2. the purposes of archaeal dna polymerase mosaic for copying template DNA, increasing or checking order of claim 1.
3. the method for copying template DNA, increasing or check order, described method comprises makes described DNA contact with the reaction mixture comprised at least:
A) the archaeal dna polymerase mosaic of claim 1;
B) damping fluid;
C) magnesium chloride;
D) the random primer of the effect being protected from exonuclease is subject to; With
E) ribonucleoside triphosphote.
4. the method for claim 3, wherein said reaction mixture also comprises polyoxyethylated sorbitan monolaurate.
5. the method for claim 3 or 4, wherein said reaction mixture also comprises ammonium salt.
6. the method any one of claim 3-4, wherein said reaction mixture also comprises sylvite.
7. the method for claim 6, wherein said sylvite is Repone K or potassium acetate.
8. the method for claim 4, the ratio of wherein said polyoxyethylated sorbitan monolaurate is the 0.003%-0.1% of total reaction volume.
9. the method for claim 8, the ratio of wherein said polyoxyethylated sorbitan monolaurate is the 0.006%-0.05% of total reaction volume.
10. the method for claim 9, the ratio of wherein said polyoxyethylated sorbitan monolaurate is the 0.01%-0.03% of total reaction volume.
The method of 11. claims 5, wherein said ammonium salt is selected from: ammonium sulfate, ammonium chloride or ammonium acetate.
The method of 12. claims 11, wherein said ammonium salt is ammonium sulfate.
The method of 13. claims 12, the concentration of wherein said ammonium sulfate is 30mM-60mM.
The method of 14. claims 13, the concentration of wherein said ammonium sulfate is 40mM-50mM.
The method of 15. claims 11, wherein said ammonium salt is ammonium chloride or ammonium acetate.
The method of 16. claims 15, the concentration of wherein said ammonium chloride or ammonium acetate is 60mM-120mM.
The method of 17. claims 16, the concentration of wherein said ammonium chloride or ammonium acetate is 80mM-100mM.
The method of 18. claims 3, wherein said damping fluid is tris-hydrochloric acid, tris-acetic acid or HEPES.
The method of 19. claims 3, the pH of wherein said damping fluid is 7.0-8.5.
The method of 20. claims 3, the concentration of wherein said magnesium chloride is 2mM-20mM.
The method of 21. claims 20, the concentration of wherein said magnesium chloride is 5mM-15mM.
The method of 22. claims 7, the concentration of wherein said Repone K or potassium acetate is 30mM-70mM.
The method of 23. claims 22, the concentration of wherein said Repone K or potassium acetate is 40mM-60mM.
The method of 24. claims 3, wherein said ribonucleoside triphosphote is dCTP, dGTP, dTTP and dATP.
The method of 25. claims 24, wherein said dCTP, dGTP, dTTP and dATP ribonucleoside triphosphote is equimolar amount.
The method of 26. claims 3, wherein said template DNA is plasmid DNA.
The method of 27. claims 3, wherein said template DNA is genomic dna.
The method of 28. claims 3, wherein said amplification is carried out under the constant temperature of 25-40 DEG C.
The method for amplification template DNA of 29. claims 3, wherein said amplification is undertaken by rolling loop type amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA) or ring mediated amplification (LAMPA).
The method of 30. claims 3, wherein at least one ribonucleoside triphosphote or a kind of primer are labeled.
31. for implementing the claims the test kit of the method any one of 3-30, and described test kit comprises:
A) the archaeal dna polymerase mosaic of claim 1;
B) damping fluid; With
C) magnesium chloride.
32. the test kit of claim 31, described test kit also comprises primer.
The test kit of 33. claims 32, described primer is random, and is subject to the effect being protected from exonuclease.
The test kit of 34. claims 33, described test kit also comprises polyoxyethylated sorbitan monolaurate.
35. the test kit of claim 33, described test kit also comprises ammonium salt.
36. the test kit of claim 33, described test kit also comprises sylvite.
37. the test kit of claim 33, described test kit also comprises ribonucleoside triphosphote.
The test kit of 38. claims 33, wherein at least one primer is labeled.
The test kit of 39. claims 37, wherein at least one ribonucleoside triphosphote is labeled.
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| PCT/ES2010/070454 WO2011000997A1 (en) | 2009-07-02 | 2010-07-01 | Phage φ 29 dna polymerase chimera |
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| EP2971080B1 (en) | 2013-03-15 | 2018-01-03 | Expedeon, S.L. | Methods for amplification and sequencing using thermostable tthprimpol |
| US20180216098A1 (en) * | 2014-08-27 | 2018-08-02 | New England Biolabs, Inc. | Synthon Formation |
| CN105483115A (en) * | 2014-09-19 | 2016-04-13 | 深圳华大基因科技有限公司 | Kit for single cell genome-wide amplification and application thereof |
| CN110268055A (en) * | 2016-12-19 | 2019-09-20 | 宽腾矽公司 | Polymerase for sequencing reaction |
| WO2020264429A1 (en) | 2019-06-28 | 2020-12-30 | Quantum-Si Incorporated | Polymerizing enzymes for sequencing reactions |
| CN110747191B (en) * | 2019-10-17 | 2021-08-17 | 深圳清华大学研究院 | Polypeptide, chimeric polymerase and application thereof |
| US20230075380A1 (en) | 2020-01-31 | 2023-03-09 | Tyris Therapeutics, S.L. | Process for the production of closed linear dna |
| EP4097253A1 (en) | 2020-01-31 | 2022-12-07 | Tyris Therapeutics, S.L. | Closed linear dna with modified nucleotides |
| EP4299731A1 (en) | 2022-06-29 | 2024-01-03 | ASOCIACIÓN CENTRO DE INVESTIGACIÓN COOPERATIVA EN NANOCIENCIAS "CIC nanoGUNE" | Synthetic primase-polymerase and uses thereof |
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| US6323009B1 (en) | 2000-06-28 | 2001-11-27 | Molecular Staging, Inc. | Multiply-primed amplification of nucleic acid sequences |
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